使用HUVEC细胞进行血管生成实验，并用LONZA核转仪转染anti-VEGFR2 siRNA
Srinivasan Kokatam1, Kanchan Tiwari1, Jenny Schroeder2, Andrea Toell2, Lubna Hussain3, Preeti Kapoor1.
1. Lonza India Pvt Ltd, Hyderabad, India; 2. Lonza Cologne GmbH, Cologne, Germany; 3. Lonza Walkersville, Inc., Walkersville, MD, U.S.A.
实验材料：
英文名 | 中文名 | 品牌 | 货号 |
single donor Clonetics™ HUVECs | 脐静脉内皮细胞，单一供体 | LONZA Clonetics | C2517A |
EGM™ 2 Media | 血管内皮细胞培养基 | LONZA | CC-3162 |
ViaLight™ Plus BioAssay Kit | 细胞活力检测试剂盒 | LONZA | LT07-221 |
Matrigel | 基质胶 | Corning | 356237 |
4D Nucleofector | 核转仪 | LONZA | C+X+96模块 |
P5 Primary Cell Nucleofector Kit | 核转试剂盒 | LONZA | V4XP-5024 V4XP-5032 |
Calcein AM | Life Technologies | C3100MP | |
VEGFR2 ELISA | VEGFR2的ELISA检测试剂盒 | R&D Systems | DYC1780-5 |
培养基配方：
实验流程简介：
1. 使用pmaxGFP质粒进行进行预实验，优化HUVEC细胞核转条件；
2. 检测核转条件对HUVEC细胞的Tube formation是否有影响；
3. 转染siRNA，确定对细胞活力影响最小的最佳浓度，确定检测蛋白表达降低的最佳时间点；
4. 评价siRNA对HUVEC细胞Tube Formation的影响。
实验结果：
图1. HUVEC细胞转染GFP质粒，转染效率约为82%，对细胞形态无明显影响：
图2. HUVEC细胞进行核转后，对Tube formation无明显影响：
图3. Anti-VEGFR2 siRNA转染后的细胞对VEGF无反应，证明siRNA发挥作用。
参考文献：
1. Timar, J. et. al. (2001) Angiogenesis-dependent diseases and angiogenesis therapy. Pathol. Oncol. Res.7 (2): 85-94
2. Murga M1, Fernandez-Capetillo O, Tosato G. (2005) Neuropilin-1 regulates attachment in human endothelial cells independently of vascular endothelial growth factor receptor-2. Blood 105 (5): 1992-9
3. Yang, S, Xin, X, Zlot, C et. al. (2001) Vascular Endothelial Cell Growth Factor-Driven Endothelial Tube Formation Is Mediated by Vascular Endothelial Cell Growth Factor Receptor-2, a Kinase Insert Domain-Containing Receptor. Arterioscler. Thromb. Vasc. Biol. 21: 1934-1940
4. Zumbansen M, Altrogge L, Toell A, Leake D, Muller-Hartmann H (2009) First siRNA Library Screening in Difficult-to-Transfect HUVEC and Jurkat Cells. White paper.
5. Zeitelhofer M, Vessey JP, Xie Y, Tubing F, Thomas S, Kiebler M, Dahm R (2007) High-efficiency transfection of mammalian neurons via Nucleofection. Nat. Protocols 2: 1692–1704
6. Kapoor, R., Reddy, V. and Kapoor, P. (2014) Tube Formation Assay with Primary Human Umbilical Vein Endothelial Cells. Resource Notes, 2014: 9-12.
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